Project Summary: The development of user-friendly devices that allow for rapid and low-cost infectious disease diagnosis are essential to combat the ever growing problem of infectious disease. Current diagnostic technologies available to clinicians often require sample processing by trained personnel in a laboratory setting and take several hours to days to produce diagnostic data to inform disease management. Recent work has demonstrated the utility of synthetic biology and cell-free synthetic gene circuits in the development precise and versatile sensor systems. These technologies have been designed to initiate gene expression to produce a detectable signal protein upon activation by a specific nucleotide or protein analyte. Currently these systems have been restricted to an optical readout of gene circuit activation, limiting the sensitivity and robustness of the developed technology. As an alternative to optical systems, electronic readout of markers of disease provides compelling simplicity, sensitivity and specificity in the detection of biological markers. A sensing system based on the marriage of synthetic biology with electrochemical readout provide a unique opportunity to harness the advantages of both technologies allowing for the development of highly versatile and sensitive technologies suitable for POC use. In this proposal we will engineer diagnostic devices based on gene switch technology allowing for the rapid detection and identification and subtyping of viral infections through an interface with electrochemical detection devices. The combination of synthetic gene circuits with electrochemical sensing will allow for more sensitive and strain-specific detection, multiplexed sensing, and rapid prototyping.! As only the gene circuits of the device will need to be altered to detect new diseases, these chips could be rapidly deployed both to traditional medical settings and the developing world to combat disease outbreaks and epidemics. In this project we will develop multiplexed sensor chips for the detection and genotyping of influenza species suitable for rapid deployment. Annual deaths from influenza infection range from 250,000 ? 500,000 with early detection and ongoing surveillance being key to patient health and containment of outbreaks, thus providing an important and relevant test case for the developed technology. In the first aim of the project we will develop and test cell-free gene circuit assay systems compatible with integration to electrochemical detection systems. In addition, electrodes capable of integration with the cell-free assays will be designed and tested to match electrode morphology with required target sensitivity. In the second aim, we will develop and validate a sensor chip capable of multiplexed electrochemical detection and validate the assay with clinical samples. The proposed study will develop and validate assays capable of sensitive and specific detection of infectious diseases. These assays will be integrated into devices for rapid, low-cost detection of disease, and are essential to combat disease outbreak.